Project Summary It is now well accepted that human gut microbiomes are actively involved in human health and that changes in our gut microbiomes from living more sanitized, industrialized, lifestyles, have led to susceptibility to certain autoimmune diseases. Our research group has made significant contributions in determining what exactly has changed during industrialization by characterizing the ancestral state of the human gut ecology. In such studies, it has become clear that industrialization has led to a decrease in gut microbiome diversity. Moreover, several strains of bacteria common to all non-industrialized, traditional, populations are missing (extirpated) in industrialized populations. In our studies, we have identified several such taxa: Prevotella, Bacteroidales S24- 7, Melainabacteria YS2, Succinivibrio, Catenibacterium and Treponema. To date, we have provided a more detailed analysis of the Treponema. Through partial genome reconstructions, we discovered there are multiple Treponema strains fitting within the evolutionary clade of bacteria that are proficient at complex carbohydrate digestion, a feature that has implications for the study of colonic health and systemic immunomodulation. Building off our previous work, the next needed step is a detailed analysis above the gene, to proteins and metabolites, as well as isolation of strains in culture. Our specific aims are thus to 1) Characterizing the diversity of extirpated taxa across traditional peoples; this includes high resolution metagenomics for communities from Burkina Faso (n=30), Uganda (n=60), and Peru (n=25), 2) Functional characterization of extirpated taxa within the gut microbiome; this incudes high resolution metaproteomic (LC-MS/MS) sequencing and fecal metabolite screening (LC/GC MS). 3) Recovery of extirpated taxa in pure culture and in vitro chemotaxonomy; this includes substrate-utilization assays to test a wide range of carbohydrate and amino acid uptake and metabolism. To our knowledge, our Aims are groundbreaking, and yet, intuitive. No such studies have been conducted on the traditional microbiome or extirpated bacteria, and yet, this is the logical next step to understand why the gut microbiome has changed in industrialized people. We are ideally poised to conduct this study. The majority of samples have already been collected, and we have ample experience in partnering with traditional peoples. Moreover, our team has experience with generating and analyzing each of these data types. These data provide a deep functional characterization to assess mechanistic, metabolic, capacities. Finally, the isolation of these bacteria in culture is an absolute necessity for downstream studies within model organisms or therapeutic applications such as probiotics. Furthermore, these isolates provide a much needed expansion to the existing gut microbe strain inventory generated by the Human Microbiome Project, which is currently biased toward organisms isolated from healthy adults from urban populations, limiting our understanding of diversity in the natural gut ecology.